Method and reagent for inhibiting influenza virus replication

ABSTRACT

An enzymatic RNA molecule which specifically cleaves an influenza virus RNA.

This application is a continuation of application Ser. No. 07/882,713, filed May 14, 1992.

BACKGROUND OF THE INVENTION

This invention relates to reagents useful as inhibitors of influenza virus replication and infection.

The following is a discussion of relevant art, none of which is admitted to be prior art to the pending claims.

Three types of influenza viruses (A, B, and C) are distinguishable by antigenic reactivities of their internal antigens. There are other biological properties which characterize the three types: (a) Influenza A viruses have been isolated from many animal species in addition to humans while influenza B and C viruses are mainly human pathogens; (b) the surface glycoproteins of influenza A exhibit much greater variability than their homologues in the B and C viruses; (c) morphological and molecular features of C viruses are distinctive from those of the A and B viruses.

The morphological characteristics of influenza viruses are a genetic trait, but spherical morphology dominates after passage in chicken embryos or tissue culture. The genes that specify morphology are uncertain, but segregate separately from the hemagglutinin (HA) and neuraminidase (NA) envelope surface proteins. Within the lipid envelope lies the matrix protein (M), which plays a structural function. Within the matrix shell are eight single-stranded RNA molecules of negative sense associated with the nucleoprotein (NP) and three large proteins (PB1, PB2, and PA) required for RNA replication and transcription. At least three viral encoded nonstructural proteins (NS1, NS2 and M2) are formed in infected cells.

The organization of the eight RNA segments within the virion has not been completely resolved. Although each segment may exist in vivo as a nucleoprotein complex, electron microscopic studies have shown that the internal component from disrupted virions is a single large helix. The virion particle does not seem to be a tight protective coat around the RNA because ribonuclease digestion of virion reduces the RNA segments to nucleotide. Genomic RNAs of influenza virus are held in a circular conformation in a virion and in infected cells by a terminal panhandle that plays a role in viral replication. The panhandle structure is present in all segments of genomic RNA.

The HA accounts for 25% of viral protein and is distributed evenly on the virion surface. It is responsible for attachment of the virus to cells and penetration of virus into cells early in infection. The HA monomer is encoded by the fourth largest RNA segment and is synthesized as a single polypeptide chain which undergoes posttranslational cleavage at a minimum of three sites. Cleavage of the HA polypeptide into HA1 and HA2 is necessary for virus particle infectivity. A sequence of 25-32 hydrophobic amino acids at the C-terminus of HA2 saves to anchor HA in the virus membrane. In spite of functional domain conservation in HA, the amino acid or nucleotide sequences of the proteins vary considerably between isolates of different subtypes.

The NA is the second subtype-specific glycoprotein of the virion and is composed of a single polypeptide chain. The NA is not evenly distributed on the surface of the virion but is found in patches. The role of the NA in the life cycle of the virus is unclear. No posttranslational cleavage of the NA polypeptide occurs. The nucleotide sequences of different NA gene isolates varies considerably between subtypes (e.g., A and B virus amino acid homology is 26-29%). The NA gene of influenza B encodes two proteins, NA and NB. The NA is thought to be structurally and functionally similar to the type A NA. The NB protein is a glycoprotein of unknown function which is 100 amino acids in length.

The nucleoprotein (NP) is one of the type-specific antigens of influenza viruses that distinguishes among the influenza type A, B, and C viruses. The NP is a multifunctional protein having a structural role in forming the nucleoprotein complex and a putative role in transcription and replication. Genetic analysis of a large number of influenza strains has revealed that the NP genes can be placed into one of five different groupings. All avian strains fall within two groups, equine strains fall within two more groups and all human and swine strains form the final group. The restriction of certain species strains to these groups suggests that the NP gene may influence species-specificity or host range.

RNA segment 7 encodes the two, M proteins (M1 and M2). The mRNA encoding M1 is colinear with RNA segment 7, whereas M2 is encoded by a spliced mRNA. The two proteins share the same initiation codon for protein synthesis and the eight amino acid residues before the 5′ splice junction of the M2 mRNA. The remaining 88 amino acids of M2 are encoded in the +1 reading frame from nucleotides 740-1104. This organization of RNA segment 7 is present in all influenza A and B viruses sequenced.

The M1 protein is a virion structural protein that is intimately associated with the lipid bilayer in close proximity to both glycoproteins and the ribonucleoprotein complex. It is also believed to have a role in the down-regulation of the virion transcriptase activity. Passively transferred monoclonal antibodies to this protein do not confer resistance to infection by influenza virus.

The M2 protein of influenza A is an integral membrane protein that is expressed at the surface of infected cells. The M2 protein may be a virion associated protein with between 14 and 68 molecules per virion. Amantadine-resistant mutants of influenza virus contain mutations in the transmembrane domain of the M2 protein. Because amantadine alters viral penetration into cells, it is likely that M2 is in the virion.

Comparison of RNA segment 7 sequences of the H3N2 (Udorn) and H1N1 (PR8) strains show that the M protein coding sequences of these viruses (isolated 38 years apart) are highly conserved. Lamb, “The genes and proteins of influenza viruses,” in. Krug. ed. The Influenza Viruses N.Y., Plenum. 1989. Comparison of 230 nucleotides of RNA segment 7 from 5 human H1N1, H2N2 and H3N2 strains isolated over a 43 year period suggests that the same segment 7 was retained throughout the antigenic shifts of HA and NA. Hall and Air, 38 J. Virol. 1, 1981.

Studies have shown that RNA segment 8 of influenza A and B encodes two nonstructural proteins which are translated from separate mRNAs. NS1 and NS2 polypeptides of influenza A share 9 amino acids at their N termini, after which the NS2 mRNA has a 423 nucleotide deletion; then, the NS2 mRNA rejoins the NS1 3′ region in the +1 reading frame. NS1 is synthesized in large amounts early in infection. NS2 is made only late in infection. Both proteins share a nuclear localization signal and can be found in the nuclei of infected cells. Large deletions occur in the carboxyl termini of the NS1 proteins of field isolates from humans or birds, which indicates that a high degree of variation can be tolerated in this polypeptide without affecting its function.

The three largest proteins of the virion (PB1, PB2 and PA) are found associated with NP and virion RNA and carry the polymerase activity which transcribes invading viral RNA. The PB1 and PB2 proteins form a complex when expressed in the absence of other virion proteins or RNA and are probably required for complementary RNA synthesis. PA and NP are required for virion RNA synthesis. The PB1 gene of influenza B virus shows 61% homology with that of the influenza A virus.

Influenza virus produces an acute febrile infection of the respiratory tract characterized by abrupt onset prominent myalgias, headache and cough. Pneumonia is the most frequent complication; it may be primary viral (due to invasion of lung parenchyma), secondary bacterial, or mixed viral and bacterial pneumonia. It may be severe and progressive or mild and segmental. Other complications which occur with less frequency include Reye's syndrome, myocarditis, pericarditis, myositis, encephalopathy and transverse myelitis. It has been estimated that the direct costs of influenza exceed $1 billion per year and may reach $3 to $5 billion. Total costs may be two to three times higher.

Two types of vaccines are available for influenza. The “split” vaccines are chemically treated to reduce pyrogenic components and are the only type given to children under 13 years of age. The “whole” vaccine is generally given to adults. Protective antibody titers are present in more than 90% of normal subjects after vaccination with influenza A antigens, but there is much less response to influenza B antigens. Additionally, elderly subjects and patients with renal failures or immunosuppression are at much greater risk to infection even with vaccination. The 70-80% efficacy of the vaccine is only observed when strain matches are good. Lower efficacy is observed when the match is not close, and when patients are immunocompromised, or in institutional situations in which virus is readily transmitted.

Two drugs, amantadine and rimantadine, are as effective as influenza vaccine in preventing influenza A infections. Unfortunately, they are not as active against influenza B, which is responsible for 20% of all influenza epidemics and in a given year may be the only virus circulating. Amantadine is approved in the United States; rimantadine is not. Both drugs appear to impair the uncoating of viral RNA in infected cells by blocking the acidification process required to open the viral particles.

Resistance to amantadine and rimantadine is easily produced in the laboratory by serial passage of strains of influenza A virus in low concentrations of the drug, and such isolates are cross-resistant to both drugs. Drug resistant strains of influenza virus are able to initiate infection of cells as effectively as their wild type progenitors. Resistance is associated with the presence of point mutations in the RNA sequence coding for the M2 protein. This occurs most frequently at amino acid 31, but may also occur at positions 27 to 34, which encompass the transmembrane domain of the protein. It has been hypothesized that M2 protein may act as an ion channel to facilitate the acidification of the virus particle, and that amantadine and rimantadine block this viral-envelope pore.

SUMMARY OF THE INVENTION

The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting influenza virus replication. Such ribozymes can be used in a method for treatment of diseases caused by these related viruses in man and other animals, including other primates. Indeed one ribozyme may be designed for treatment of many of the diseases caused by these viruses.

Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro. Kim et al., 84 Proc. Nat. Acad. of Sci. USA 8788, 1987, Haseloff and Gertaek, 334 Nature 585, 1988, Cech, 260 JAMA 3030, 1988, and Jefferies et al., 17 Nucleic Acid Research 1371, 1989.

Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA.

This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.

Many regions of the RNAs associated with influenza viruses are appropriate targets for ribozyme attack. A ribozyme targeted to these regions in influenza viruses may be useful for targeting an equivalent region in related viruses whose sequences are unknown.

There is a high degree of nucleotide sequence homology within the sequences of the human and non-human influenza virus genomes and mRNAs. Particularly useful regions include sequences in the 5′ and 3′ regions of the genomic RNA segments and complementary regions at the 3′ ends of all influenza A and B virus mRNAs and their homologous positions in other genomes. In addition, the 3′ sequence which contributes to the panhandle (CCUGCUUUUGCU) is highly conserved among all influenza virus isolates and contains two putative ribozyme cleavage sites in a relatively accessible region of the RNA. The panhandle sequence (AGUAGAAACAAGG) at the 5′ ends of the RNAs also contains a suitable ribozyme target site. The complementary sequence (CCUUGUUUCUACU) of the 5′ panhandle is present at the 3′ terminus in all mRNAs and contains an additional 4 ribozyme target sites which occur within accessible regions of these molecules (i.e., the loosely base-paired terminal regions). Detailed information of the sequence of the eight RNA segments of influenza (seven in influenza C strains) and the molecular weights of the encoded proteins is known.

Ribozymes targeting any of the above regions of these genomes should be able to cleave the RNAs in a manner which will inhibit the translation of the molecules.

Thus, in the first aspect the invention features an enzymatic RNA molecule (or ribozyme) which specifically cleaves influenza virus RNA or its complementary mRNA.

Preferred cleavage is at regions required for viral replication (e.g., protein synthesis, such as the regions in RNA segment 7 which regulate or encode the M proteins) the conserved 5′ and 3′ regions of the genomic RNA segments which are involved in the panhandle structures, as well as the regions at the 3′ ends of all mRNAs which are complementary to the 5′ panhandle structure and their equivalent in other viruses. Alternative regions may also be used as targets for ribozyme-mediated cleavage of these viral genomes. Each target can be chosen as described below, e.g., by a study of the secondary structure of the RNA, and the individual role of such RNA in the replication of the virus. If the targets are contained within the open reading frames of regions which encode proteins essential to the replication of the virus, then these other targets are preferred candidates for cleavage by ribozymes, and subsequent inhibition of viral replication. The regions encoding the influenza A virus PB1, PB2 and PA proteins and their homologous proteins in the other viruses are examples of such preferred targets.

By “enzymatic RNA molecule” or by “catalytic RNA molecule” it is meant an ENA molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic RNA molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. By “equivalent” RNA to influenza virus is meant to include those naturally occurring RNA molecules associated with viral caused diseases in various animals, including humans, and other primates. These viral RNAs have similar structures and equivalent genes to each other.

In preferred embodiments, the enzymatic RNA molecule is formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are described by Rossi et al., 8 AIDS RESEARCH AND HUMAN RETROVIRUSES 183, 1992, of hairpin motifs by Hampel et al., RNA CATALYST FOR CLEAVING SPECIFIC RNA SEQUENCES, filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, 28 Biochemistry 4929, 1989 and Hampel et al., 18 Nucleic Acids Research 299, 1990, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 31 Biochemistry 16, 1992, of the RNaseP motif by Guerrier-Takada et al., 35 Cell 849, 1983, and of the group I intron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic RNA molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

In particularly preferred embodiments, the RNA which is cleaved in influenza virus RNA is selected from one or more of the following sequences:

genomic RNAs CCUGCUUUUGCU (Seq. ID. No. 1) AGUAGAAACAAGG (Seq. ID. No. 2) all mRNAs CCUUGUUUCUACU (Seq. ID. No. 3) M protein mRNAs nu- cleo- tide number   8 GCAGGUAGAUAUUGAAAGATGAG (Seq. ID. No. 4)  35 CUAACCGAGG (Seq. ID. No. 5)  64 UAUCGUCCCGUCAGGCC (Seq. ID. No. 6)  81 CCCUCAAAGCCGAGAUCGCG (Seq. ID. No. 7) 159 GGCUAAAGACA (Seq. ID. No. 8) 266 CAAAAUGCCCUAAAUGGGAAUGGAG (Seq. ID. No. 9) 312 CAGUCAAACUAUACAGGAAACUG (Seq. ID. No. 10) 331 ACUGAAAAGAGAGAUAA (Seq. ID. No. 11) 433 AACGGUAACCACA (Seq. ID. No. 12) 466 GUGUGCCACUUG (Seq. ID. No. 13) 512 AGACAGAUGGUAACUACUACC (Seq. ID. No. 14) 537 CACUAAUAAGGCAUGAAAACAG (Seq. ID. No. 15) 556 CAGAAUGGUGCUG (Seq. ID. No. 16) 578 ACGGCUAAGGCUAUGGAGCAG (Seq. ID. No. 17) 626 GAACGCAUGG (Seq. ID. No. 18) 652 UAGGCAGAUGGUGCAGGCGAUGAGG (Seq. ID. No. 19) 671 AUGAGGACUAUUGGGACUCACCC (Seq. ID. No. 20) 691 CCCUAGCUCCAGUG (Seq. ID. No. 21) 739 GGCCUACCAAAAACGGAUGGGAGUG (Seq. ID. No. 22) 783 GAUCCUCUCAUUAUUGCC (Seq. ID. No. 23) 825 UUGAUAUUG (Seq. ID. No. 24) 840 CUUGAUCGUC (Seq. ID. No. 25) 863 UAUUUAUCGUCGCCUUAAAUA (Seq. ID. No. 26) 902 UUCUACGGAAGGAGUGCCU (Seq. ID. No. 27) 921 GAGUCUAUGAGGGA (Seq. ID. No. 28) 938 GUAUCGGCAGGAACAACA (Seq. ID. No. 29) 951 CAACAGAGUGUAGUGG (Seq. ID. No. 30) 977 UGGUCAUUUU (Seq. ID. No. 31) 995 AGAGCUGGAGUAAAAACUACCUUG (Seq. ID. No. 32)

In a second related aspect, the invention features a vertebrate cell which includes an enzymatic RNA molecule as described above. Preferably, the vertebrate man or other primate cell.

In a third related aspect, the invention features an expression vector which includes nucleic acid encoding the enzymatic RNA molecules described above, located in the vector, e.g., in a manner which allows expression of that enzymatic RNA molecule within a vertebrate cell.

In a fourth related aspect, the invention features a method for treatment of a influenza virus-caused fisease by administering to a patient an enzymatic RNA molecule which cleaves influenza virus RNA, e.g., in the 5′ panhandle region.

The invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the viral RNA of influenza virus in virus-infected cells or virion particles. The ribozyme molecule is preferably targeted to a highly conserved sequence region of an influenza virus such that all types and strains of these viruses can be treated with a single ribozyme. Such enzymatic RNA molecules can be delivered exogenously to infected cells. In the preferred hammerhead motif the small size (less than 40 nucleotides, preferably between 32 and 36 nucleotides in length) of the molecule allows the cost of treatment to be reduced compared to other ribozyme motifs.

Synthesis of ribozymes greater than 100 nucleotides in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. Delivery of ribozymes by expression vectors is primarily feasible using only ex vivo treatments. This limits the utility of this approach. In this invention, small ribozyme motifs (e.g., of the hammerhead structure, shown generally in FIG. 1) are used for exogenous delivery. The simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure. Thus, unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-ribozyme flanking sequences to interfere with correct folding of the ribozyme structure or with its complementary region.

The enzymatic RNA molecules of this invention can be used to treat influenza virus infections. Infected animals can be treated at the time of productive infection. This timing of treatment will reduce viral loads in infected cells and disable viral replication in any subsequent rounds of infection. This is possible because the preferred ribozymes disable those structures required for successful initiation of viral protein synthesis.

The preferred targets of the present invention are advantageous over other targets since they do not act only at the time of viral absorption or genomic replication during infection. In addition, viral particles which are released during a first round of infection in the presence of such ribozymes will still be immunogenic by virtue of having their virions intact. Thus, one method of this invention allows the creation of defective but immunogenic viral particles, and thus a continued possibility of initiation of an immune response in a treated animal.

In addition, the enzymatic RNA molecules of this invention can be used in vitro in a cell culture infected with influenza viruses to produce viral particles which have intact capsids and defective genomic RNA. These particles can then be used for instigation of immune responses in a prophylactic manner, or as a treatment of infected animals.

In yet another aspect, the invention features use of influenza viruses as vectors for carrying an enzymatic RNA molecule to a cell infected with another virus. Such vectors can be formed by standard methodology.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawing will first briefly be described.

Drawing

FIG. 1 is a diagrammatic representation of a hammerhead motif ribozyme showing stems I, II and III (marked (I), (II) and (III) respectively) interacting with a virus target region. The 5′ and 3′ ends of both ribozyme and target are shown. Dashes indicate base-paired nucleotides.

Target Sites

The genome of influenza viruses may be subject to rapid genetic drift by virtue of its RNA content and the nature of errors in genomic replication. Those regions (genes) of the genome which are essential for virus replication, however, are expected to maintain a constant sequence (i.e., are conserved) over extensive periods of time. These regions are preferred target sites in this invention since they are more likely to be conserved between different types or strains of influenza viruses, and thus only one ribozyme is needed to destroy all such viruses. Thus, one ribozyme may be used to target all influenza viruses. We have selected several such RNA regions of these viruses, and examined their nucleotide sequences for the presence of conserved areas which may be cleaved by ribozymes targeted to those regions. Three regions analyzed in detail are the 5′ and 3′ panhandle regions, the complementary 3′ regions present in all mRNAs, and the mRNAs encoding the M proteins; other genes can be analyzed in a manner similar to that described below.

Ribozymes targeting selected regions of the influenza virus genome are preferably chosen to cleave the target RNA in a manner which inhibits expression of the RNA. Genes are selected such that viral replication is inhibited, e.g., by inhibiting protein synthesis or genomic replication and packaging into virions. Selection of effective target sites within these critical regions of viral RNA entails testing the accessibility of the target RNA to hybridization with various oligonucleotide probes. These studies can be performed using RNA probes and assaying accessibility by cleaving the hybrid molecule with RNaseH (see below). Alternatively, such a study can use ribozyme probes designed from secondary structure predictions of the RNAs, and assaying cleavage products by polyacrylamide gel electrophoresis (PAGE), to detect the presence of cleaved and uncleaved molecules.

The following is but one example of a method by which suitable target sites can be identified and is not limiting in this invention. Generally, the method involves identifying potential cleavage sites for a hammerhead ribozyme, and then testing each of these sites to determine their suitability as targets by ensuring that secondary structure formation is minimal.

The genomic sequences of the viruses are compared in their potential target regions. Putative ribozyme cleavage sites are found to be highly conserved between strains and species of virus sequence. These sites represent the preferred sites for hammerhead or other ribozyme cleavage within these target RNAs.

Short RNA substrates corresponding to each of the gene sites are designed. Each substrate is composed of two to three nucleotides at the 5′ and 3′ ends that will not base pair with a corresponding ribozyme recognition region. The unpaired regions flank a central region of 12-14 nucleotides to which complementary arms in the ribozyme are designed.

The structure of each substrate sequence is predicted using a PC fold computer program. Sequences which give a positive free energy of binding are accepted. Sequences which give a negative free energy are modified by trimming one or two bases from each of the ends. If the modified sequences are still predicted to have a strong secondary structure, they are rejected.

After substrates are chosen, ribozymes are designed to each of the RNA substrates. Ribozyme folding is also analyzed using PC fold.

Ribozyme molecules are sought which form hammerhead motif stem II (see FIG. 1) regions and contain flanking arms which are devoid of intramolecular base pairing. Often the ribozymes are modified by trimming a base from the ends of the ribozyme, or by introducing additional base pairs in stem II to achieve the desired fold. Ribozymes with incorrect folding are rejected. After substrate/ribozyme pairs are found to contain correct intramolecular structures, the molecules are folded together to predict intermolecular interactions. A schematic representation of a ribozyme with its coordinate base pairing to its cognate target sequence is shown in FIG. 1.

Using such analyses, predictions of effective target sites in the viral genes, based upon computer generated sequence comparisons, were obtained. These are identified as SEQ. ID. NOS. 1-32, shown above.

Those targets thought to be useful as ribozyme targets can be tested to determine accessibility to nucleic acid probes in a ribonuclease H assay (see below). This assay provides a quick test of the use of the target site without requiring synthesis of a ribozyme. It can be used to screen for sites most suited for ribozyme attack.

Synthesis of Ribozymes

Ribozymes useful in this invention can be produced by gene transcription as described by Cech, supra, or by chemical synthesis. Chemical synthesis of RNA is similar to that for DNA synthesis. The additional 2′-OH group in RNA, however, requires a different protecting group strategy to deal with selective 3′-5′ internucleotide bond formation, and with RNA susceptibility to degradation in the presence of bases. The recently developed method of RNA synthesis utilizing the t-butyldimethylsilyl group for the protection of the 2′ hydroxyl is the most reliable method for synthesis of ribozymes. The method reproducibly yields RNA with the correct 3′-5′ internucleotide linkages, with average coupling yields in excess of 99%, and requires only a two-step deprotection of the polymer.

A method based upon H-phosphonate chemistry gives a relatively lower coupling efficiency than a method based upon phosphoramidite chemistry. This is a problem for synthesis of DNA as well. A promising approach to scale-up of automatic oligonucleotide synthesis has been described recently for the H-phosphonates. A combination of a proper coupling time and additional capping of “failure” sequences gave high yields in the synthesis of oligodeoxynucleotides in scales in the range of 14 micromoles with as little as 2 equivalents of a monomer in the coupling step. Another alternative approach is to use soluble polymeric supports (e.g., polyethylene glycols), instead of the conventional solid supports. This method can yield short oligonucleotides in hundred milligram quantities per batch utilizing about 3 equivalents of a monomer in a coupling step.

Various modifications to ribozyme structure can be made to enhance the utility of ribozymes. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such ribozymes to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Exogenous delivery of ribozymes benefits from chemical modification of the backbone, e.g., by the overall negative charge of the ribozyme molecule being reduced to facilitate diffusion across the cell membrane. The present strategies for reducing the oligonucleotide charge include: modification of internucleotide linkages by ethylphosphonates, use of phosphoramidites, linking oligonucleotides to positively charged molecules, and creating complex packages composed of oligonucleotides, lipids and specific receptors or effectors for targeted cells. Examples of such modifications include sulfur-containing ribozymes containing phosphorothioates and phosphorodithioates as internucleotide linkages in RNA. Synthesis of such sulfur-modified ribozymes is achieved by use of the sulfur-transfer reagent, ³H-1,2-benzenedithiol-3-one 1,1-dioxide. Ribozymes may also contain ribose modified ribonucleotides. Pyrimidine analogues are prepared from uridine using a procedure employing diethylamino sulphur trifluoride (DAST) as a starting reagent. Ribozymes can also be either electrostatically or covalently attached to polymeric cations for the purpose of reducing charge. The polymer can be attached to the ribozyme by simply converting the 3′-end to a ribonucleoside dialdehyde which is obtained by a periodate cleavage of the terminal 2′,3′-cis diol system. Depending on the specific requirements for delivery systems, other possible modifications may include different linker arms containing carboxyl, amino or thiol functionalities. Yet further examples include use of methylphosphonates and 2′-O-methylribose and 5′ or 3′ capping or blocking with m₇GpppG or m₃ ^(2,2,7)GpppG.

For example, a kinased ribozyme is contacted with guanosine triphosphate and guanyltransferase to add a m³G cap to the ribozyme. After such synthesis, the ribozyme can be gel purified using standard procedure. To ensure that the ribozyme has the desired activity, it may be tested with and without the 5′ cap using standard procedures to assay both its enzymatic activity and its stability.

Synthetic ribozymes, including those containing various modifiers, can be purified by high pressure liquid chromatography (HPLC). Other liquid chromatography techniques, employing reverse phase columns and anion exchangers on silica and polymeric supports may also be used.

There follows an example of the synthesis of one ribozyme. A solid phase phosphoramidite chemistry is employed. Monomers used are 2′-tert-butyl-dimethylsilyl cyanoethylphosphoramidities of uridine, N-benzoyl-cytosine, N-phenoxyacetyl adenosine and guanosine (Glen Research, Sterling, Va.). Solid phase synthesis is carried out on either an ABI 394 or 380B DNA/RNA synthesizer using the standard protocol provided with each machine. The only exception is that the coupling step is increased from 10 to 12 minutes. The phosphoramidite concentration is 0.1 M. Synthesis is done on a 1 μmole scale using a 1 μmole RNA reaction column (Glen Research). The average coupling efficiencies are between 97% and 98% for the 394 model, and between 97% and 99% for the 380B model, as determined by a calorimetric measurement of the released trityl cation.

Blocked ribozymes are cleaved from the solid support (e.g., CPG), and the bases and diphosphoester moiety deprotected in a sterile vial by dry ethanolic ammonia (2 mL) at 55° C. for 16 hours. The reaction mixture is cooled on dry ice. Later, the cold liquid is transferred into a sterile screw cap vial and lyophilized.

To remove the 2′-tert-butyl-dimethylsilyl groups from the ribozyme, the residue is suspended in 1 M tetra-n-butylammonium fluoride in dry THF (TBAF), using a 20 fold excess of the reagent for every silyl group, for 16 hours at ambient temperature (about 15-25° C.). The reaction is quenched by adding an equal volume of sterile 1 M triethylamine acetate, pH 6.5. The sample is cooled and concentrated on a SpeedVac to half the initial volume.

The ribozymes are purified in two steps by HPLC on a C4 300 Å 5 mm DeltaPak column in an acetonitrile gradient.

The first step, or “trityl on” step, is a separation of 5′-DMT-protected ribozyme(s) from failure sequences lacking a 5′-DMT group. Solvents used for this step are: A (0.1 M triethylammonium acetate, pH 6.8) and B (acetonitrile). The elution profile is: 20% B for 10 minutes, followed by a linear gradient of 20% B to 50% B over 50 minutes, 50% B for 10 minutes, a linear gradient of 50% B to 100% B over 10 minutes, and a linear gradient of 100% B to 0% B over 10 minutes.

The second step is a purification of a completely deblocked ribozyme by a treatment of 2% trifluoroacetic acid on a C4 300 Å 5 mm DeltaPak column in an acetonitrile gradient. Solvents used for this second step are: A (0.1 M Triethylammonium acetate, pH 6.8) and B (80% acetonitrile, 0.1 M triethylammonium acetate, pH 6.8). The elution profile is: 5% B for 5 minutes, a linear gradient of 5% B to 15% B over 60 minutes, 15% B for 10 minutes, and a linear gradient of 15% B to 0% B over 10 minutes.

The fraction containing ribozyme is cooled and lyophilized on a SpeedVac. Solid residue is dissolved in a minimum amount of ethanol and sodium perchlorate in acetone. The ribozyme is collected by centrifugation, washed three times with acetone, and lyophilized.

Expression Vector

While synthetic ribozymes are preferred in this invention, those produced by expression vectors can also be used. In designing a suitable ribozyme expression vector the following factors are important to consider. The final ribozyme must be kept as small as possible to minimize unwanted secondary structure within the ribozyme. A promoter (e.g., the human cytomegalovirus immediate early promoter) should be chosen to be a relatively strong promoter, and expressible both in vitro and in vivo. Such a promoter should express the ribozyme at a level suitable to effect production of enough ribozyme to destroy a target RNA, but not at too high a level to prevent other cellular activities from occurring (unless cell death itself is desired).

A hairpin at the 5′ end of the ribozyme is useful to ensure that the required transcription initiation sequence (GG or GGG or GGGAG) does not bind to some other part of the ribozyme and thus affect regulation of the transcription process. The 5′ hairpin is also useful to protect the ribozyme from 5′-3′ exonucleases. A selected hairpin at the 3′ end of the ribozyme is useful since it acts as both a transcription termination signal, and as a protection from 3′-5′ exonucleases. One example of a known termination signal is that present on the T7 RNA polymerase system. This signal is about 30 nucleotides in length. Other 3′ hairpins of shorter length can be used to provide good termination and RNA stability. Such hairpins can be inserted within the vector sequences to allow standard ribozymes to be placed in an appropriate orientation and expressed with such sequences attached.

Poly(A) tails are also useful to protect the 3′ end of the ribozyme. These can be provided by either including a poly(A) signal site in the expression vector (to signal a cell to add the poly(A) tail in vivo), or by introducing a poly(A) sequence directly into the expression vector. In the first approach the signal must be located to prevent unwanted secondary structure formation with other parts of the ribozyme. In the second approach, the poly(A) stretch may reduce in size over time when expressed in vivo, and thus the vector may need to be checked over time. Care must be taken in addition of a poly(A) tail which binds poly(A) binding proteins which prevent the ribozyme from acting upon their target sequence.

Ribozyme Testing

Once the desired ribozymes are selected, synthesized and purified, they are tested in kinetic and other experiments to determine their utility. An example of such a procedure is provided below.

Preparation of Ribozyme

Crude synthetic ribozyme (typically 350 μg at a time) is purified by separation on a 15% denaturing polyacrylamide gel (0.75 mm thick, 40 cm long) and visualized by UV shadowing. Once excised, gel slices containing full length ribozyme are soaked in 5 ml gel elution buffer (0.5 M NH₄OAc, 1 mM EDTA) overnight with shaking at 4° C. The eluent is desalted over a C-18 matrix (Sep-Pak cartridges, Millipore, Milford, Mass.) and vacuum dried. The dried RNA is resuspended in 50-100 μl TE (TRIS 10 mM, EDTA 1 mM, pH 7.2). An aliquot of this solution is diluted 100 fold into 1 ml TE, half of which is used to spectrophotometrically quantitate the ribozyme solution. The concentration of this dilute stock is typically 150-800 nM. Purity of the ribozyme is confirmed by the presence of a single band on a denaturing polyacrylamide gel.

A ribozyme may advantageously be synthesized in two or more portions. Each portion of a ribozyme will generally have only limited or no enzymatic activity, and the activity will increase substantially (by at least 5-10 fold) when all portions are ligated (or otherwise juxtaposed) together. A specific example of hammerhead ribozyme synthesis is provided below.

The method involves synthesis of two (or more) shorter “half” ribozymes and ligation of them together using T4 RNA ligase. For example, to make a 34 mer ribozyme, two 17 mers are synthesized, one is phosphorylated, and both are gel purified. These purified 17 mers are then annealed to a DNA splint strand complementary to the two 17 mers. This DNA splint has a sequence designed to locate the two 17 mer portions with one end of each adjacent each other. The juxtaposed RNA molecules are then treated with T4 RNA ligase in the presence of ATP. The 34 mer RNA so formed is then HPLC purified.

Preparation of Substrates

Approximately 10-30 pmoles of unpurified substrate is radioactively 5′ end-labelled with T4 polynucleotide kinase using 25 pmoles of [γ-³²P] ATP. The entire labelling mix is separated on a 20% denaturing polyacrylamide gel and visualized by autoradiography. The full length band is excised and soaked overnight at 4° C. in 100 μl of TE (10 mM Tris-HCl pH 7.6, 0.1 mM EDTA).

Kinetic Reactions

For reactions using short substrates (between 8 and 16 bases) a substrate solution is made 1X in assay buffer (75 mM Tris-HCl₁, pH 7.6; 0.1 mM EDTA, 10 MM MgCl₂) such that the concentration of substrate is less than 1 nM. A ribozyme solution (typically 20 nM) is made 1X in assay buffer and four dilutions are made using 1X assay buffer. Fifteen μl of each ribozyme dilution (i.e., 20, 16, 12, 8 and 4 nM) is placed in a separate tube. These tubes and the substrate tube are pre-incubated at 37° C. for at least five minutes.

The reaction is started by mixing 15 μl of substrate into each ribozyme tube by rapid pipetting (note that final ribozyme concentrations are 10, 8, 6, 4, 2 nM). 5 μl aliquots are removed at 15 or 30 second intervals and quenched with 5 μl stop solution (95% formamide, 20 mM EDTA xylene cyanol, and bromphenol blue dyes). Following the final ribozyme time point, an aliquot of the remaining substrate is rmoved as a zero ribozyme control.

The samples are separated on either 15% or 20% polyacrylamide gels. Each gel is visualized and quantitated with an Ambis beta scanner (Ambis Systems, San Diego, Calif.).

For the most active ribozymes, kinetic analyses are performed in substrate excess to determine K_(m) and K_(cat) values.

For kinetic reactions with long RNA substrates (greater than 15 bases in length) the substrates are prepared by transcription using T7 RNA polymerase and defined templates containing a T7 promoter, and DNA encoding appropriate nucleotides of the viral RNA. The substrate solution is made lx in assay buffer (75 mM Tris-HCl, pH 7.6; 0.1 mM EDTA; 10 mM MgCl₂) and contains 58 nanomolar concentration of the long RNA molecules. The reaction is started by addition of gel purified ribozymes to 1 μM concentration. Aliquots are removed at 20, 40, 60, 80 and 100 minutes, then quenched by the addition of 5 μl stop solution. Cleavage products are separated using denaturing PAGE. The bands are visualized and quantitated with an Ambis beta scanner.

Kinetic Analysis

A simple reaction mechanism for ribozyme-mediated cleavage is: ${R + S}\underset{k_{- 1}}{\overset{k_{1}}{\underset{\rightarrow}{\leftarrow}}}\left\lbrack {R\text{:}S} \right\rbrack \overset{k_{2}}{\underset{\rightarrow}{\leftarrow}}{\left\lbrack {R\text{:}P} \right\rbrack \begin{matrix} \left. \rightarrow{R + P} \right. \end{matrix}}$

where R=ribozyme, S=substrate, and P=products. The boxed step is important only in substrate excess. Because ribozyme concentration is in excess over substrate concentration, the concentration of the ribozyme-substrate complex ([R:S]) is constant over time except during the very brief time when the complex is being initially formed, i.e.,: $\frac{\left\lbrack {R\text{:}S} \right\rbrack}{t} = 0$

where t=time, and thus:

(R) (S)k ₁=(RS) (k ₂ +k ₁).

The rate of the reaction is the rate of disappearance of substrate with time: ${Rate} = {\frac{- {(S)}}{t} = {k_{2}\left( {R\quad S} \right)}}$

Substituting these expressions: ${(R)(S)k_{1}} = {{1/k_{2}}\frac{- {(S)}}{t}\left( {k_{2} + k_{1}} \right)}$ ${\text{or:}\quad \frac{- {(S)}}{S}} = {\frac{k_{1}k_{2}}{\left( {k_{2} + k_{1}} \right)}(R){t}}$

Integrating this expression with.respect to time yields: ${{- \ln}\quad \frac{S}{S_{0}}} = {\frac{k_{1}k_{2}}{\left( {k_{2} + k_{1}} \right)}(R)\quad t}$

where S₀=initial substrate. Therefore, a plot of the negative log of fraction substrate uncut versus time (in minutes) yields a straight line with slope: ${slope} = {{\frac{k_{1}k_{2}}{\left( {k_{2} + k_{1}} \right)}(R)} = k_{obs}}$

where k_(obs)=observed rate constant. A plot of slope (k_(obs)) versus ribozyme concentration yields a straight line with a slope which is: ${slope} = {\frac{k_{1}k_{2}}{\left( {k_{2} + k_{1}} \right)}\quad {which}\quad {is}\quad \frac{k_{cat}}{K_{m}}}$

Using these equations the data obtained from the kinetic experiments provides the necessary information to determine which ribozyme tested is most useful, or active. Such ribozymes can be selected and tested in in vivo or ex vivo systems. An example of preparing ribozymes in a liposome delivery vehicle is given below.

Liposome Preparation

Lipid molecules are dissolved in a volatile organic solvent (CHCl₃, methanol, diethylether, ethanol, etc.). The organic solvent is removed by evaporation. The lipid is hydrated into suspension with 0.lx phosphate buffered saline (PBS), then freeze-thawed 3x using liquid nitrogen and incubation at room temperature. The suspension is extruded sequentially through a 0.4 μm, 0.2 μm and 0.1 μm polycarbonate filters at maximum pressure of 800 psi. The ribozyme is mixed with the extruded liposome suspension and lyophilized to dryness. The lipid/ribozyme powder is rehydrated with water to one-tenth the original volume. The suspension is diluted to the minimum volume required for extrusion (0.4 ml for 1.5 ml barrel and 1.5 ml for 10 ml barrel) with 1xPBS and re-extruded through 0.4 μm, 0.2 μm, 0.1 μm polycarbonate filters. The liposome entrapped ribozyme is separated from untrapped ribozyme by gel filtration chromatography (SEPHAROSE CL-4B, BIOGEL A5M). The liposome extractions are pooled and sterilized by filtration through a 0.2 μm filter. The free ribozyme was pooled and recovered by ethanol precipitation. The liposome concentration is determined by incorporation of a radioactive lipid. The ribozyme concentration is determined by labeling with ³²P. Rossi et al., 1992 supra (and references cited therein) describe other methods suitable for preparation of liposomes.

In Vivo Assay

The efficacy of action of a chosen ribozyme may be tested in vivo by use of cell cultures sensitive to a selected influenza virus, using standard procedures. For example, monolayer cultures of virus-sensitive cells are grown in 6 or 96 well tissue culture plates. Prior to infection with influenza virus, cultures are treated for 3 to 24 hours with ribozyme-containing liposomes. Cells are then rinsed with phosphate buffered saline (PBS) and virus added at a multiplicity of 1-100 pfu/cell. After a one-hour adsorption, free virus is rinsed away using PBS, and the cells are treated for three to five days with appropriate liposome preparations and medium changes. Virus is harvested from cells into the overlying medium. Cells are broken by three cycles of incubation at −70° C. and 37° C. for 30 minutes at each temperature, and viral titers determined by plaque assay using established procedures. These procedures can be modified for each specific virus to be tested.

Ribonuclease Protection Assay

The accumulation of target mRNA in cells or the cleavage of the RNA by ribozymes or RNaseH (in vitro or in vivo) can be quantified using an RNase protection assay.

In this method, antisense riboprobes are transcribed from template DNA using T7 RNA polymerase (U.S. Biochemicals) in 20 μl reactions containing 1X transcription buffer (supplied by the manufacturer), 0.2 mM ATP, GTP and UTP, 1 U/μl pancreatic RNase inhibitor (Boehringer Mannheim Biochemicals) and 200 μCi ³²P-labeled CTP (800 Ci/mmol, New England Nuclear) for 1 h at 37° C. Template DNA is digested with 1 U RNase-free DNase I (U.S. Biochemicals, Cleveland, Ohio) at 37° C. for 15 minutes and unincorporated nucleotides removed by G-50 SEPHADEX spin chromatography.

In a manner similar to the transcription of antisense probe, the target RNA can be transcribed in vitro using a suitable DNA template. The transcript is purified by standard methods and digested with ribozyme at 37° C. according to methods described later.

Alternatively, virus-infected cells are harvested into 1 ml of PBS, transferred to a 1.5 ml EPPENDORF tube, pelleted for 30 seconds at low speed in a microcentrifuge, and lysed in 70 μl of hybridization buffer (4 M guanidine isothiocyanate, 0.1% sarcosyl, 25 mM sodium citrate, pH 7.5). Cell lysate (45 μl) or defined amounts of in vitro transcript (also in hybridization buffer) is then combined with 5 μl of hybridization buffer containing 5×10⁵ cpm of each antisense riboprobe in 0.5 ml EPPENDORF tubes, overlaid with 25 μl mineral oil, and hybridization accomplished by heating overnight at 55° C. The hybridization reactions are diluted into 0.5 ml RNase solution (20 U/ml RNase A, 2 U/ml RNase T1, 10 U/ml RNase-free DNAse I in 0.4 M NaCl), heated for 30 minutes at 37° C., and 10 μl of 20% SDS and 10 μl of Proteinase K (10 mg/ml) added, followed by an additional 30 minutes incubation at 37° C. Hybrids are partially purified by extraction with 0.5 ml of a 1:1 mixture of phenol/chloroform; aqueous phases are combined with 0.5 ml isopropanol, and RNase-resistant hybrids pelleted for 10 minutes at room temperature (about 20° C.) in a microcentrifuge. Pellets are dissolved in 10 μl loading buffer (95% formamide, 1X TBE, 0.1% bromophenol blue, 0.1% xylene cylanol), heated to 95° C. for five minutes, cooled on ice, and analyzed on 4% polyacrylamide/7 M urea gels under denaturing conditions.

Ribozyme Stability

The chosen ribozyme can be tested to determine its stability, and thus its potential utility. Such a test can also be used to determine the effect of various chemical modifications (e.g., addition of a poly(A) tail) on the ribozyme stability and thus aid selection of a more stable ribozyme. For example, a reaction mixture contains 1 to 5 pmoles of 5′ (kinased) and/or 3′ labeled ribozyme, 15 μg of cytosolic extract and 2.5 mM MgCl₂ in a total volume of 100 μl. The reaction is incubated at 37° C. Eight μl aliquots are taken at timed intervals and mixed with 8 μl of a stop mix (20 mM EDTA, 95% formamide). Samples are separated on a 15% acrylamide sequencing gel, exposed to film, and scanned with an Ambis.

A 3′-labelled ribozyme can be formed by incorporation of the ³²P-labeled cordycepin at the 3′ OH using poly(A) polymerase. For example, the poly(A) polymerase reaction contains 40 mM Tris, pH 8, 10 mM MgCl₂, 250 mM NaCl, 2.5 mM MnCl₂; 3 μl P³² cordycepin, 500 Ci/mM; and 6 units poly(A) polymerase in a total volume of 50 μl. The reaction mixture is incubated for 30 minutes at 37° C.

Effect of Base Substitution Upon Ribozyme Activity

To determine which primary structural characteristics could change ribozyme cleavage of substrate, minor base changes can be made in the substrate cleavage region recognized by a specific ribozyme. For example, the substrate sequences can be changed at the central “C” nucleotide, changing the cleavage site from a GUC to a GUA motif. The K_(cat)/K_(m) values for cleavage using each substrate are then analyzed to determine if such a change increases ribozyme cleavage rates. Similar experiments can be performed to address the effects of changing bases complementary to the ribozyme binding arms. Changes predicted to maintain strong binding to the complementary substrate are preferred. Minor changes in nucleotide content can alter ribozyme/substrate interactions in ways which are unpredictable based upon binding strength alone. Structures in the catalytic core region of the ribozyme recognize trivial changes in either substrate structure or the three dimensional structure of the ribozyme/substrate complex.

To begin optimizing ribozyme design, the cleavage rates of ribozymes containing varied arm lengths, but targeted to the same length of short RNA substrate can be tested. Minimal arm lengths are required and effective cleavage varies with ribozyme/substrate combinations.

The cleavage activity of selected ribozymes can be assessed using picornavirus RNA substrates. The assays are performed in ribozyme excess and approximate K_(cat)/K_(min) values obtained. Comparison of values obtained with short and long substrates indicates utility in vivo of a ribozyme.

Intracellular Stability of Liposome-delivered Ribozymes

To test the stability of a chosen ribozyme in vivo the following test is useful. Ribozymes are ³²P-end labeled, entrapped in liposomes and delivered to influenza virus sensitive cells for three hours. The cells are fractionated and purified by phenol/chloroform extraction. Cells (1×10⁷, T-175 flask) are scraped from the surface of the flask and washed twice with cold PBS. The cells are homogenized by douncing 35 times in 4 ml of TSE (10 mM Tris, pH 7.4, 0.25 M Sucrose, mM EDTA). Nuclei are pelleted at 100xg for 10 minutes. Subcellular organelles (the membrane fraction) are pelleted at 200,000xg for two hours using an SW60 rotor. The pellet is resuspended in 1 ml of H buffer (0.25 M Sucrose, 50 mM HEPES, pH 7.4). The supernatant contains the cytoplasmic fraction (in approximately 3.7 ml). The nuclear pellet is resuspended in 1 ml of 65% sucrose in TM (50 mM Tris, pH 74., 2.5 mM MgCl₂) and banded on a sucrose step gradient (1 ml nuclei in 65% sucrose TM, 1 ml 60% sucrose TM, 1 ml 55% sucrose TM, 50% sucrose TM, 300 ul 25% sucrose TM) for one hour at 37,000xg with an SW60 rotor. The nuclear band is harvested and diluted to 10% sucrose with TM buffer. Nuclei are pelleted at 37,000xg using an SW60 rotor for 15 minutes and the pellet resuspended in 1 ml of TM buffer. Aliquots are size fractionated on denaturing polyacrylamide gels and the intracellular localization determined. By comparison to the migration rate of newly synthesized ribozyme, the various fraction containing intact ribozyme can be determined.

To investigate modifications which would lengthen the half-life of ribozyme molecules intracellularly, the cells may be fractioned as above and the purity of each fraction assessed by assaying enzyme activity known to exist in that fraction.

The various cell fractions are frozen at −70° C. and used to determine relative nuclease resistances of modified ribozyme molecules. Ribozyme molecules may be synthesized with 5 phosphorothioate (ps), or 2′-O-methyl (2′-OMe) modifications at each end of the molecule. These molecules and a phosphodiester version of the ribozyme are end-labeled with ³²P and ATP using T4 polynucleotide kinase. Equal concentrations are added to the cell cytoplasmic extracts and aliquots of each taken at 10 minute intervals. The samples are size fractionated by denaturing PAGE and relative rates of nuclease resistance analyzed by scanning the gel with an Ambis β-scanner. The results show whether the ribozymes are digested by the cytoplasmic extract, and which versions are relatively more nuclease resistant. Modified ribozymes generally maintain 80-90% of the catalytic activity of the native ribozyme when short RNA substrates are employed.

Unlabeled, 5′ end-labeled or 3′ end-labeled ribozymes can be used in the assays. These experiments can also be performed with human cell extracts to verify the observations.

Administration of Ribozyme

Selected ribozymes can be administered prophylactically, or to virus infected patients, e.g., by exogenous delivery of the ribozyme to an infected tissue by means of an appropriate delivery vehicle, e.g., a liposome, a controlled release vehicle, by use of iontophoresis, electroporation or ion paired molecules, or covalently attached adducts, and other pharmacologically approved methods of delivery. Routes of administration include intramuscular, aerosol, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal. Expression vectors for immunization with ribozymes and/or delivery of ribozymes are also suitable.

The specific delivery route of any selected ribozyme will depend on the use of the ribozyme. Generally, a specific delivery program for each ribozyme will focus on naked ribozyme uptake with regard to intracellular localization, followed by demonstration of efficacy. Alternatively, delivery to these same cells in an organ or tissue of an animal can be pursued. Uptake studies will include uptake assays to evaluate cellular oligonucleotide uptake, regardless of the delivery vehicle or strategy. Such assays will also determine the intracellular localization of the ribozyme following uptake, ultimately establishing the requirements for maintenance of steady-state concentrations within the cellular compartment containing the target sequence (nucleus and/or cytoplasm). Efficacy and cytotoxicity can then be tested. Toxicity will not only include cell viability but also cell function.

Some methods of delivery that may be used include:

a. encapsulation in liposomes,

b. transduction by retroviral vectors,

c. conjugation with cholesterol,

d. localization to nuclear compartment utilizing antigen binding site found on most snRNAs,

e. neutralization of charge of ribozyme by using nucleotide derivatives, and

f. use of blood stem cells to distribute ribozymes throughout the body.

At least three types of delivery strategies are useful in the present invention, including: ribozyme modifications, particle carrier drug delivery vehicles, and retroviral expression vectors. Unmodified ribozymes, like most small molecules, are taken up by cells, albeit slowly. To enhance cellular uptake, the ribozyme may be modified essentially at random, in ways which reduces its charge but maintains specific functional groups. This results in a molecule which is able to diffuse across the cell membrane, thus removing the permeability barrier.

Modification of ribozymes to reduce charge is just one approach to enhance the cellular uptake of these larger molecules. The random approach, however, is not advisable since ribozymes are structurally and functionally more complex than small drug molecules. The structural requirements necessary to maintain ribozyme catalytic activity are well understood by those in the art. These requirements are taken into consideration when designing modifications to enhance cellular delivery. The modifications are also designed to reduce susceptibility to nuclease degradation. Both of these characteristics should greatly improve the efficacy of the ribozyme. Cellular uptake can be increased by several orders of magnitude without having to alter the phosphodiester linkages necessary for ribozyme cleavage activity.

Chemical modifications of the phosphate backbone will reduce the negative charge allowing free diffusion across the membrane. This principle has been successfully demonstrated for antisense DNA technology. The similarities in chemical composition between DNA and RNA make this a feasible approach. In the body, maintenance of an external concentration will be necessary to drive the diffusion of the modified ribozyme into the cells of the tissue. Administration routes which allow the diseased tissue to be exposed to a transient high concentration of the drug, which is slowly dissipated by systemic adsorption are preferred. Intravenous administration with a drug carrier designed to increase the circulation half-life of the ribozyme can be used. The size and composition of the drug carrier restricts rapid clearance from the blood stream. The carrier, made to accumulate at the site of infection, can protect the ribozyme from degradative processes.

Drug delivery vehicles are effective for both systemic and topical administration. They can be designed to serve as a slow release reservoir, or to deliver their contents directly to the target cell. An advantage of using direct delivery drug vehicles is that multiple molecules are delivered per uptake. Such vehicles have been shown to increase the circulation half-life of drugs which would otherwise be rapidly cleared from the blood stream. Some examples of such specialized drug delivery vehicles which fall into this category are liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.

From this category of delivery systems, liposomes are preferred. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity.

Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. They have an internal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter. Several studies have shown that liposomes can deliver RNA to cells and that the RNA remains biologically active.

For example, a liposome delivery vehicle originally designed as a research tool, Lipofectin, has been shown to deliver intact mRNA molecules to cells yielding production of the corresponding protein.

Liposomes offer several advantages: They are non-toxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues. Finally, cost effective manufacture of liposome-based pharmaceuticals, either in a liquid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivery system.

Other controlled release drug delivery systems, such as nonoparticles and hydrogels may be potential delivery vehicles for a ribozyme. These carriers have been developed for chemotherapeutic agents and protein-based pharmaceuticals, and consequently, can be adapted for ribozyme delivery.

Topical administration of ribozymes is advantageous since it allows localized concentration at the site of administration with minimal systemic adsorption. This simplifies the delivery strategy of the ribozyme to the disease site and reduces the extent of toxicological characterization. Furthermore, the amount of material to be applied is far less than that required for other administration routes. Effective delivery requires the ribozyme to diffuse into the infected cells. Chemical modification of the ribozyme to neutralize negative charge may be all that is required for penetration. However, in the event that charge neutralization is insufficient, the modified ribozyme can be co-formulated with permeability enhancers, such as Azone or oleic acid, in a liposome. The liposomes can either represent a slow release presentation vehicle in which the modified ribozyme and permeability enhancer transfer from the liposome into the infected cell, or the liposome phospholipids can participate directly with the modified ribozyme and permeability enhancer in facilitating cellular delivery. In some cases, both the ribozyme and permeability enhancer can be formulated into a suppository formulation for slow release.

Ribozymes may also be systemically administered. Systemic absorption refers to the accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include: intravenous, subcutaneous, intraperitoneal, intranasal, intrathecal and ophthalmic. Each of these administration routes expose the ribozyme to an accessible diseased tissue. Subcutaneous administration drains into a localized lymph node which proceeds through the lymphatic network into the circulation. The rate of entry into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier localizes the ribozyme at the lymph node. The ribozyme can be modified to diffuse into the cell, or the liposome can directly participate in the delivery of either the unmodified or modified ribozyme to the cell.

A liposome formulation which can deliver ribozymes to lymphocytes and macrophages is also useful for the initial site of influenza virus replication is in tissues of the nasopharynx and respiratory system. Coating of lymphocytes with liposomes containing ribozymes will target the ribozymes to infected cells expressing viral surface antigens. Whole blood studies show that the formulation is taken up by 90% of the lymphocytes after 8 hours at 37° C. Preliminary biodistribution and pharmacokinetic studies yielded 70% of the injected dose/gm of tissue in the spleen after one hour following intravenous administration.

Intraperitoneal administration also leads to entry into the circulation, with once again, the molecular weight or size controlling the rate of entry.

Liposomes injected intravenously show accumulation in the liver, lung and spleen. The composition and size can be adjusted so that this accumulation represents 30% to 40% of the injected dose. The rest is left to circulate in the blood stream for up to 24 hours.

The chosen method of delivery should result in cytoplasmic accumulation and molecules should have some nuclease-resistance for optimal dosing. Nuclear delivery may be used but is less preferable. Most preferred delivery methods include liposomes (10-400 nm), hydrogels, controlled-release polymers, microinjection or electroporation (for ex vivo treatments) and other pharmaceutically applicable vehicles. The dosage will depend upon the disease indication and the route of administration but should be between 100-200 mg/kg of body weight/day. The duration of treatment will extend through the course of the disease symptoms, usually at least 14-16 days and possibly continuously. Multiple daily doses are anticipated for topical applications, ocular applications and vaginal applications. The number of doses will depend upon disease delivery vehicle and efficacy data from clinical trials.

Establishment of therapeutic levels of ribozyme within the cell is dependent upon the rate of uptake and degradation. Decreasing the degree of degradation will prolong the intracellular half-life of the ribozyme. Thus, chemically modified ribozymes, e.g., with modification of the phosphate backbone, or capping of the 5′ and 3′ ends of the ribozyme with nucleotide analogs may require different dosaging. Descriptions of useful systems are provided in the art cited above, all of which is hereby incorporated by reference herein.

The claimed ribozymes are also useful as diagnostic tools to specifically or non-specifically detect the presence of a target RNA in a sample. That is, the target RNA, if present in the sample, will be specifically cleaved by the ribozyme, and thus can be readily and specifically detected as smaller RNA species. The presence of such smaller RNA species is indicative of the presence of the target RNA in the sample.

Other embodiments are within the following claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 32 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCUGCUUUUG CU 12 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2: AGUAGAAACA AGG 13 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCUUGUUUCU ACU 13 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCAGGUAGAU AUUGAAAGAT GAG 23 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CUAACCGAGG 10 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6: UAUCGUCCCG UCAGGCC 17 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCCUCAAAGC CGAGAUCGCG 20 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGCUAAAGAC A 11 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CAAAAUGCCC UAAAUGGGAA UGGAG 25 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CAGUCAAACU AUACAGGAAA CUG 23 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ACUGAAAAGA GAGAUAA 17 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AACGGUAACC ACA 13 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GUGUGCCACU UG 12 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AGACAGAUGG UAACUACUAC C 21 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CACUAAUAAG GCAUGAAAAC AG 22 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CAGAAUGGUG CUG 13 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ACGGCUAAGG CUAUGGAGCA G 21 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAACGCAUGG 10 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 19: UAGGCAGAUG GUGCAGGCGA UGAGG 25 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 20: AUGAGGACUA UUGGGACUCA CCC 23 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CCCUAGCUCC AGUG 14 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GGCCUACCAA AAACGGAUGG GAGUG 25 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GAUCCUCUCA UUAUUGCC 18 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 24: UUGAUAUUG 9 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 25: CUUGAUCGUC 10 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 26: UAUUUAUCGU CGCCUUAAAU A 21 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 27: UUCUACGGAA GGAGUGCCU 19 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GAGUCUAUGA GGGA 14 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GUAUCGGCAG GAACAACA 18 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CAACAGAGUG UAGUGG 16 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 31: UGGUCAUUUU 10 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 32: AGAGCUGGAG UAAAAAACUA CCUUG 25 

What is claimed is:
 1. A catalytic RNA molecule which is active to specifically cleave the sequence shown as any of SEQ. ID. NOS. 1-32.
 2. The catalytic RNA molecule of claim 1 wherein said RNA molecule is in a hammerhead motif.
 3. The catalytic RNA molecule of claim 1 wherein said RNA molecule is in a hairpin, hepatitis Delta virus, group 1 intron, or RNaseP RNA motif.
 4. A vertebrate cell comprising a catalytic RNA molecule of any of claims 1, 2 or
 3. 5. The cell of claim 4, wherein said cell is a human cell.
 6. An expression vector comprising nucleic acid encoding the catalytic RNA molecule of any of claims 1, 2 or 3, in a manner which allows expression of that catalytic RNA molecule within a vertebrate cell.
 7. An influenza virus comprising an catalytic RNA molecule.
 8. The influenza virus of claim 7 wherein said catalytic RNA molecule forms part of the genomic RNA of said influenza virus. 